Immunological agglutination reactions are used for identifying blood types and detecting various antibodies and antigens in blood samples and other aqueous medium.
In a conventional procedure, particles with binding agents, such as red blood cells, are mixed with a sample or reagent in test tubes or microtiter plates, and the mixture may then be incubated and centrifuged. Various reactions either occur or do not occur depending on antigens or antibodies present in the particle surface and reagent sample. Typically, these reactions manifest themselves as clumps of cells or particles, referred to as agglutinates. Thus, the absence of such clumps indicates that no reaction has occurred; and the presence of such clumps indicates that a reaction has occurred, with the size and amount of such clumps being a semi-quantitative indicator of the level or concentration of antigens or antibodies in the sample, or an indicator of the reaction strength, affinity of the complex for which the blood sample was tested.
Recently, a new agglutination assay, referred to as column agglutination technology (CAT), has been developed. This agglutination testing method utilizes filtration as a means of separating agglutinated particles from non-reactive components for immunoassay applications. In this method, gel or glass bead microparticles are contained in a small column, referred to as microcolumn, along with a reagent such as Anti-IgG. Red blood cells, or particles with binding agents, are placed in a reaction chamber above the column. During centrifugation, the cells or particles are mixed with reagent and may react in the column. If the reaction occurs, part or all of the cells are agglutinated and trapped in the bead area after centrifugation. If the reaction does not occur, the non-agglutinated cells are forced toward the bottom of the column by the centrifugal force. As a result, the nature and distribution of the particles in the microcolumn after centrifugation provides a visual indication of whether any reaction occurred, and if so, of the strength of the reaction.
Conventionally, an agglutination reaction is classified as negative (if no reaction occurred) or as positive (if a reaction has occurred) and if positive, the reaction is further classified as a class of +0.5, +1, +2, +3 or +4, depending on the strength of the antigen-antibody interaction. Indeterminate reaction is given if the nature of the reaction cannot be surely classified. In the CAT method, the classes of agglutination reactions can be determined on the basis of red cell distribution pattern in the microcolumn.
In U.S. Pat. No. 5,594,808, there is disclosed a system and software for automatically classifying the types of agglutinate reactions described in the previous paragraph, referred to as normal reactions. That system and software works well in most instances. Occasionally, however, there are other types of immunological reactions with red blood cells, which are considered abnormal for the purposes of this invention. These include:
Hemolysis reaction: In a hemolysis reaction, part or all of red blood cells are broken (hemolyzed) due to the antigen-antibody reaction. Once the cells are broken, the hemoglobin in the red blood cells are released into the test sample, resulting in the change of liquid color to red.
Mixed field reaction: In a mixed field reaction, part of red blood cells are agglutinated while the remaining red cells do not agglutinate. This may indicate that the test sample contains two different population of red cells, which may be caused by a previous transfusion or other pathological conditions.
These reactions and other abnormalities can interfere with detection by the system and software described in the aforesaid '808 patent.